A look into the Pyrenophora teres f. teres colonization strategies on barley using a transformation-free staining and confocal microscope analysis

Authors: NELSON, ASHLEY - North Dakota State University; KARIYAWASAN, GAYAN - North Dakota State University; Wyatt, Nathan; STUKENBROCK, EVA - Max Planck Institute For Evolutionary Biology; BOROWICZ, PAWEL - North Dakota State University; LIU, ZHAOHUI - North Dakota State University; Friesen, Timothy

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 12/6/2023
Publication Date: 3/12/2024
Citation: Nelson, A., Kariyawasan, G., Wyatt, N.A., Stukenbrock, E., Borowicz, P., Liu, Z., Friesen, T.L. 2024. A look into the Pyrenophora teres f. teres colonization strategies on barley using a transformation-free staining and confocal microscope analysis. Meeting Abstract.

Interpretive Summary:

Technical Abstract: Laser scanning confocal microscopy is an invaluable tool in assessing plant microbe interactions at a cellular level. Here we use a transformation-free staining technique with propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. Showcasing this, in tandem with the fungal pathogen Pyrenophora teres f. teres (Ptt) infecting barley, we show how high-resolution images shed light on fungal colonization strategies and infection structures of fungal pathogens. In the Ptt-barley interaction, intracellular vesicles develop in epidermal cells directly below penetration points and serve as branching points for hyphal growth into the plant’s mesophyll layer. Infected plant mesophyll layers are full of deliberate intercellular hyphal growth that maximizes its surface areas to grow around the individual mesophyll cells, exhibiting patterns we characterize as encasement, mesophyll cell trapping, thick layering, and branching. Encasement is the growth of hyphae in the mesophyll where it surrounds the cells on two opposing sides. Mesophyll cell trapping begins as encasement, but the hyphae continue to grow around the whole mesophyll cells surrounding it on all sides. Thick layering is the layered parallel growth of multiple hyphae and branching is the perpendicular growth of hyphae through numerous layers of mesophyll cells. We analyzed morphological differences between avirulent and virulent isogenic strains of Ptt and used the growth patterns mentioned above to assess their success in-planta. Hyphae of virulent strains were most intent on growing parallel to the length of the leaf, through the mesophyll layer as rapidly as possible, followed by lateral branching, explaining the net like lesions characteristic of this disease. Cell death was only observed behind the growing point of the fungus, where mesophyll cells were surrounded by the fungal hyphae. Comparatively, the avirulent isogenic isolate was able to grow in-planta but had a fitness deficiency that inhibited its quick takeover of the leaf tissue. We believe the pathogen is maximizing fungal biomass to absorb nutrients at a high efficiency while delaying plant defenses before cell death is an advantage to the pathogen. Ptt has shown the potential of this technique to relook at the strategies of fungal pathogens and work in tandem with quantitative and molecular analysis.